*Target (protein/gene name): microcin immunity protein MccF *NCBI Gene # or RefSeq#:821575425 *Protein ID (NP or XP #) or Wolbachia#:BAR77315 *Organism (including strain): Bacillus anthracis str. Ames Etiologic Risk Group (see link below): */Disease Information (sort of like the Intro to your Mini Research Write up): Bacillus anthracis, more commonly known as anthrax, a bacteria normally found in the soil that can transform itself into a spore to survive years under the harshest conditions. Once these spores are in favorable conditions, they germinate and become thriving colonies and gain the ability to produce toxins which is what makes the bacteria such a potent killer in humans as well as animals. Link to TDR Targets page (if present):n/a Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.) Essentiality of this protein: http://www.ncbi.nlm.nih.gov/protein/BAR77315.1
Is it a monomer or multimer as biological unit? (make prediction athttp://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Multimer Complex of proteins?: No Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
yes with SELENOMETHIONINE *EC#: 4.2.1.11 Link to BRENDA EC# page:brenda-enzymes.org/enzyme.php?ecno=4.2.1.11 --Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): Expression organism: E. coli (BL21(DE3) magic Media Type: M9 minimal Media volume: 5mL Growth Temp: 37*C Induction Temp: 18*C Induction Reagent: 1 mM IPTG Experiment type: assay
Current Inhibitors:There are 18 compounds Expression Information (has it been expressed in bacterial cells):It can be expressed in E. coli BL21 (DE3) Purification Method: Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): >3GJZ:A|PDBID|CHAIN|SEQUENCE
*length of your protein in Amino Acids:336 Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website-37984.4 Molar Extinction coefficient of your protein at 280 nm wavelength: 52830 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it. *CDS Gene Sequence (paste as text only):
1 mstiidvyar evldsrgnpt vevevytesg afgraivpsg astgeheave lrdgdksryl 61 gkgvmnavnn vneaiapeiv gfdvtdqagi dramieldgt pnkgklgana ilgvsmavah 121 aaadfvglpl yrylggfnak qlptpmmnii nggshadnnv dfqefmilpv gaptfkesir 181 mgaevfhalk avlhdkglnt avgdeggfap nlgsnreale viieaiekag ykagenvflg 241 mdvassefyn ketgkydlag egrtgltsae mvdfyeelck dfpiisiedg ldendwdghk 301 llterigdkv qlvgddlfvt ntqklaegie kgisnsilik vnqigtltet feaiemakra 361 gytavvshrs getedatiad iavatnagqi ktgsmsrtdr iakynqllri edelgeiavy 421 dgiksfynik r *GC% Content for gene: *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
microcin immunity protein MccF
*NCBI Gene # or RefSeq#: 821575425
*Protein ID (NP or XP #) or Wolbachia#: BAR77315
*Organism (including strain):
Bacillus anthracis str. Ames
Etiologic Risk Group (see link below):
*/Disease Information (sort of like the Intro to your Mini Research Write up):
Bacillus anthracis, more commonly known as anthrax, a bacteria normally found in the soil that can transform itself into a spore to survive years under the harshest conditions. Once these spores are in favorable conditions, they germinate and become thriving colonies and gain the ability to produce toxins which is what makes the bacteria such a potent killer in humans as well as animals.
Link to TDR Targets page (if present): n/a
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.)
Essentiality of this protein:
http://www.ncbi.nlm.nih.gov/protein/BAR77315.1
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Multimer
Complex of proteins?: No
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
yes with SELENOMETHIONINE
*EC#: 4.2.1.11
Link to BRENDA EC# page:brenda-enzymes.org/enzyme.php?ecno=4.2.1.11
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
Expression organism:
E. coli (BL21(DE3) magic
Media Type: M9 minimal
Media volume: 5mL
Growth Temp: 37*C
Induction Temp: 18*C
Induction Reagent: 1 mM IPTG
Experiment type: assay
-- link to Sigma (or other company) page for assay (see Sigma links below)
http://www.csgid.org/cake/expressions/view/12879
-- -or link (or citation) to paper that contains assay information
-- links to assay reagents (substrates) pages.
IPTG- http://www.sigmaaldrich.com/catalog/product/sial/i6758?lang=en®ion=US
E. coli- http://www.sigmaaldrich.com/catalog/product/sigma/ge27154201?lang=en®ion=US
M9 Minimal solution- http://www.sigmaaldrich.com/catalog/product/sigma/m9956?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #
IPTG - $61.40 for 1G (16758)
E. coli BL21 - $236.50 for 1 vial (GE27-1542-01)
M9 Minimal Salts, 5x - $47.70 for 500mL (M9956)
All supplied by Sigma-Aldrich
Structure (PDB or Homology model)
Current Inhibitors: There are 18 compounds
Expression Information (has it been expressed in bacterial cells): It can be expressed in E. coli BL21 (DE3)
Purification Method:
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
>3GJZ:A|PDBID|CHAIN|SEQUENCE
SNAMPLPKSLKYGDTIGIYSPSSPVTYTSPKRFERAKSYLLQKGFHILEGSLTGRYDYYRSGSIQERAKELNALIRNPNV
SCIMSTIGGMNSNSLLPYIDYDAFQNNPKIMIGYSDATALLLGIYAKTGIPTFYGPALVPSFGEFEPFVDDTYKYFLETL
LHDQALPYNIKQPLFWSDEFINWEEKTKEKELRPNNWISVTNGQATGRVIGGNLNTIQGIWGSPYMPCIQEGDILFIEDS
SKDAATIERSFSFLKINGVFDKVSGIILGKHEQFDDCGTNRKPYEILLEVLQNQRIPLLADFDCCHTHPMITMPIGVQVK
MDATNKTIHILEKWKI
*length of your protein in Amino Acids: 336
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website- 37984.4
Molar Extinction coefficient of your protein at 280 nm wavelength: 52830
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
1 mstiidvyar evldsrgnpt vevevytesg afgraivpsg astgeheave lrdgdksryl
61 gkgvmnavnn vneaiapeiv gfdvtdqagi dramieldgt pnkgklgana ilgvsmavah
121 aaadfvglpl yrylggfnak qlptpmmnii nggshadnnv dfqefmilpv gaptfkesir
181 mgaevfhalk avlhdkglnt avgdeggfap nlgsnreale viieaiekag ykagenvflg
241 mdvassefyn ketgkydlag egrtgltsae mvdfyeelck dfpiisiedg ldendwdghk
301 llterigdkv qlvgddlfvt ntqklaegie kgisnsilik vnqigtltet feaiemakra
361 gytavvshrs getedatiad iavatnagqi ktgsmsrtdr iakynqllri edelgeiavy
421 dgiksfynik r
*GC% Content for gene:
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):